The 'Layering Hypothesis,' first proposed in by Herzenberg in 1989, theorized that there are lineages of lymphocytes arising at different stages of development. A basal stratum of so-called innate lymphocytes, the B1 cells and ?? T cells, was proposed to provide a first line of immune defense as well as other functions such as maintenance of tissue homeostasis. Recent data from mouse and human strongly support the 'layering hypothesis,' but little is known of such lineages over evolutionary time. The immunoglobulin (Ig) gene organization in cartilaginous fish (sharks, skates, and rays), the so-called cluster organization, allows for plasticity in the types of genes that can be selected and used in different ways; for example, some Ig heavy chain clusters have been selected for adaptive immunity with single-domain variable regions and others are 'germline-joined,' meaning that the rearranging gene segments were joined by RAG in germ cells and perpetuated in the population as fixed genes. One particular germline-joined heavy chain makes up over half of the serum Ig in neonates, and consistent with what has been shown in mice, the early antibody recognizes apoptotic cells, suggesting that it is involved in homeostatic functions. We will continue to study the structure and function of this molecule, concentrating on its ligand specificity. This IgM is expressed in plasma cells as a first wave during development, followed by at least two other waves of plasma cells. The 2nd wave expresses exclusively a multimeric form of IgM (19S) and is identified as J chain- positive/BLIMP1-negative, and the 3rd wave is J chain-negative/BLIMP1-positive (7S). The dichotomy in BLIMP1 and J chain expression suggests that it might be used as a universal marker for plasma cell lineages, and we plan to test this proposal in mouse and Xenopus, representatives of two other highly divergent vertebrate taxa. We will also examine the structure and function of ?? T cell receptors in Xenopus and sharks, two species in which immunoglobulin variable regions are used in a large proportion of the ? chains. Based on this finding, as well as uncovering such Ig/TCR chimeras in many other vertebrates, suggests that there are subpopulations of adaptive ?? T cell receptors in all vertebrates, which has not been appreciated. The study of this system will not only uncover layers of ?? T cell and B cell development in the oldest animals with adaptive immunity based on Ig/TCR/MHC, but we hypothesize that it may serve as a simple paradigm for layering of lymphocyte lineages in all other vertebrates.